Project Summary: The candidate, Donna L. Cioffi, is a first-year postdoctoral fellow in the laboratory of Dr. Troy Stevens at the University of South Alabama (USA). The candidate's immediate career goals include two additional years at USA, as a postdoc, performing the first phase of the proposed project. Long-term career goals include a tenure-track position at a university. Although the candidate's husband is a tenured faculty member at USA, she is not limiting her search to just USA. The candidate and her husband are willing to relocate to a new university should an opportunity arise for both. During the mentored phase of the project the candidate will be trained in patch clamp electrophysiology and basic molecular biology techniques. These techniques will be used throughout the mentored and independent phases of the proposed project in addition to future studies. This research project focuses on endothelial barrier disruption, a contributing factor to the development and progression of pathophysiological states including inflammation, atherosclerosis and acute respiratory distress syndrome. The endothelial Isoc channel is a calcium selective ion channel whose activation is an important step leading to the formation of inter-endothelial cell gaps and endothelial barrier disruption. Isoc activation/inactivation properties largely determine the amount of calcium which enters the cell and as such, influence the magnitude and dynamics of gap formation. A protein 4.1 binding domain together with an adjacent proline-rich region located on the TRPC4 subunit of the Isoc channel are important for channel activation. In the mentored phase of this project, the independent roles of the protein 4.1 binding domain and proline-rich region in channel activation or inactivation will be determined. Further, while it is known that Isoc inactivation is both calcium- and phosphorylation-dependent, it is unknown whether these pathways are part of a common mechanism. The independent phase of this project will determine whether a common calcium/phosphate pathway underlies inactivation of the Isoc channel.